Downloaded from molecularcasestudies.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press COLD SPRING HARBOR Molecular Case Studies | RESEARCH ARTICLE Recurrent SPECC1L–NTRK fusions in pediatric sarcoma and brain tumors Dong-Anh Khuong-Quang,1,2,3 Lauren M. Brown,1,3,4 Marie Wong,1,5 Chelsea Mayoh,1 Alexandra Sexton-Oates,3 Amit Kumar,1,6 Mark Pinese,1 Sumanth Nagabushan,7 Loretta Lau,1,7 Louise E. Ludlow,3 Andrew J. Gifford,1,8 Michael Rodriguez,8 Jayesh Desai,6,9 Stephen B. Fox,9,10 Michelle Haber,1 David S. Ziegler,1,7 Jordan R. Hansford,2,3,4 Glenn M. Marshall,1,7 Mark J. Cowley,1,5 and Paul G. Ekert1,3,5,6 1Children’s Cancer Institute, University of New South Wales, Randwick, 2031, Australia; 2Children’s Cancer Centre, 3Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, 3052, Australia; 4Department of Pediatrics, University of Melbourne, Parkville, 3052, Australia; 5School of Women’s and Children’s Health, UNSW Medicine, UNSW Sydney, Randwick, 2031, Australia; 6Peter MacCallum Cancer Centre, Melbourne, 3000, Australia; 7Kids Cancer Centre, Sydney Children’s Hospital, Randwick, 2031, Australia; 8Department of Anatomical Pathology, Prince of Wales Hospital, Randwick, 2031, Australia; 9Sir Peter MacCallum Department of Oncology, University of Melbourne, 3000, Australia; 10Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, 3000, Australia Abstract The identification of rearrangements driving expression of neurotrophic receptor tyrosine kinase (NTRK) family kinases in tumors has become critically important because of the availability of effective, specific inhibitor drugs. Whole-genome sequencing (WGS) combined with RNA sequencing (RNA-seq) can identify novel and recurrent expressed fu- sions. Here we describe three SPECC1L–NTRK fusions identified in two pediatric central nervous system cancers and an extracranial solid tumor using WGS and RNA-seq. These fu- sions arose either through a simple balanced rearrangement or in the context of a complex chromoplexy event. We cloned the SPECC1L–NTRK2 fusion directly from a patient sample and showed that enforced expression of this fusion is sufficient to promote cytokine-inde- pendent survival and proliferation. Cells transformed by SPECC1L–NTRK2 expression are sensitive to a TRK inhibitor drug. We report here that SPECC1L–NTRK fusions can arise Corresponding author: in a range of pediatric cancers. Although WGS and RNA-seq are not required to detect [email protected] NTRK fusions, these techniques may be of benefit when NTRK fusions are not suspected on clinical grounds or not identified by other methods. © 2020 Khuong-Quang et al. This article is distributed under the terms of the Creative Commons [Supplemental material is available for this article.] Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided INTRODUCTION that the original author and source are credited. Genomic and transcriptomic sequencing of pediatric cancers has broadened the spectrum of driver genetic alterations recognized across diverse tumor types. A subset of these drivers Ontology term: neoplasm of the is targetable with novel therapies, which in some cases provide clinical options when none central nervous system previously existed. The TRK family of receptors, encoded by the NTRK1, NTRK2,and NTRK3 genes, are proto-oncogenes regulating signaling pathways, including the MAPK Published by Cold Spring Harbor and PI3K pathways, and play a critical role in neuronal development and differentiation. Laboratory Press Fusions that link protein–protein interaction domains to the tyrosine kinase domain of each doi:10.1101/mcs.a005710 of the NTRK genes are recognized oncogenic drivers. NTRK fusions were first described in Cite this article as Khuong-Quang et al. 2020 Cold Spring Harb Mol Case Stud 6: a005710 1 of 17 Downloaded from molecularcasestudies.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press COLD SPRING HARBOR Molecular Case Studies Recurrent SPECC1L–NTRK fusions colorectal carcinoma (Martin-Zanca et al. 1986) and papillary thyroid carcinoma (Bongarzone et al. 1989) and later in a broad range of cancer types including pediatric cancers (Knezevich et al. 1998; Davis et al. 2018). NTRK fusions are strongly associated with particular rare pedi- atric tumor types, notably infantile fibrosarcoma (Knezevich et al. 1998), in which 70%–90% of cases harbor recurrent ETV6–NTRK3 fusions (Bourgeois et al. 2000; Sheng et al. 2001). NTRK fusions also characterize a subset of more common tumors, such as pediatric gliomas (Mackay et al. 2017; Guerreiro Stucklin et al. 2019; Clarke et al. 2020). The clinical identification of NTRK fusions is a high priority because of the availability and clinical efficacy of TRK inhibitors (Drilon et al. 2018). The dramatic antitumoral activity of TRK inhibitors appears to be independent of the clinical features of the tumor in which it arises, including histological type, patient age, and genomic mechanisms generating the fusion (Drilon et al. 2018; Laetsch et al. 2018). Larotrectinib is only the second agent to be granted U.S. Federal Drug Administration (FDA) approval based on the presence of a molecular al- teration, regardless of the type of cancer in which the mutation occurs, and a trial in previous- ly untreated pediatric solid tumors is underway (COG ADVL1823—NCT03834961). To emphasize the importance of detecting a potential NTRK fusion, the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group has recommended testing for NTRK fusions in all advanced stage malignancies, particularly if standard testing has not identified clear driver mutations (Marchio et al. 2019). Whole-genome sequencing (WGS) and whole-transcriptome RNA sequencing (RNA- seq) offer an unbiased methodology to identify the presence of all expressed structural variants such as NTRK fusions. NTRK fusions, either recurrent, rare, or with unknown fusion partners can also be detected with a range of next-generation sequencing (NGS) approach- es, including panels sequencing RNA after library preparation based on hybridization cap- ture or anchored multiplex polymerase chain reaction (PCR) (Seager et al. 2019). More specific and sensitive genetic techniques including fluorescence in situ hybridization (FISH) and PCR-based assays are efficient and rapid but are limited to known fusions and risk missing unique or rare recurrent NTRK fusions. Immunohistochemical techniques detect TRK overexpression as a surrogate for the presence of a driver fusion but have limitations in specificity, particularly for NTRK3 fusions (Penault-Llorca et al. 2019). Here, we report the genomic features of three instances of a recurrent NTRK fusion part- ner, SPECC1L, in diverse pediatric cancer subtypes. The mechanisms giving rise to these fu- sions vary from simple reciprocal translocations to very complex chromosomal events resembling chromoplexy. We have cloned and expressed the SPECC1L–NTRK2 fusion and show that it is sufficient to permit cytokine-independent survival and proliferation. These data emphasize the significant potential clinical benefit of performing WGS and RNA-seq to identify recurrent targetable lesions in difficult-to-treat tumors in which driver le- sions have not been identified by conventional testing. RESULTS Clinical Presentation Table 1 summarizes the clinical details of each of the three patients. Patient 1 presented with neurological symptoms of ataxia and intermittent headaches and was diagnosed at the age of 10 yr with an unusual pineal low-grade neuroepithelial tumor. The tumor had two morpho- logically distinct components. One contained a moderate density of mildly pleomorphic, strongly GFAP-positive, Olig2- and CD34-negative glial cells with extensive psammomatous calcification. The other component was more cellular, containing plump spindle-shaped and large epithelioid cells with occasional perivascular pseudorosettes resembling astroblastom- atous pseudorosette. Most of these cells were also strongly GFAP-positive, although some of Khuong-Quang et al. 2020 Cold Spring Harb Mol Case Stud 6: a005710 2 of 17 Downloaded from molecularcasestudies.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press COLD SPRING HARBOR Molecular Case Studies Recurrent SPECC1L–NTRK fusions Table 1. Summary of patient characteristics Gender M M F Age 10 yr 16 mo 11 mo Clinical Three weeks history of Epilepsy and focal Failure to thrive, presentation dizziness, ataxia, and neurological deficit developmental regression, intermittent headaches and intermittent fevers; acute respiratory distress Diagnosis Low-grade Anaplastic astrocytoma Infantile fibrosarcoma neuroepithelial tumor Primary tumor Pineal Left thalamus Chest Staging Localized Localized Localized Histopathology Low-grade Anaplastic astrocytoma Congenital infantile sarcoma neuroepithelial tumor Ki67 staining Ki67 2%–3% Ki67 10% Ki67 10% Pretreatment Surgery alone Carboplatin-based infant VCR/Cyclo/ActD followed by regimen (five cycles VCR before tumor progression) Other reportable — Homozygous loss of — findings CDKN2A/B Treatment Surveillance Larotrectinib Larotrectinib Status SD 22 mo postsurgery SD on larotrectinib for CR postsurgery after 22 mo 33 mo on larotrectinib (M) Male, (F) female, (VCR) vincristine, (Cyclo) cyclophosphamide, (ActD) dactinomycin, (FC) fold change, (SD) stable disease, (CR) complete remission. the epithelioid